The presence of molecules in or on cell are responsible for a variety of effects. For example, various abnormalities appear to be the result of the undesired expression of a particular protein. Thus, many tumors are believed to be the result of the overexpression of cellular oncogenes, such as neu, myc, abl, etc. Other malignancies are believed to be the result of expression of an altered receptor. Still other illnesses, such as certain leukaemias, result from the overexpression of cellular receptors which leads to the unregulated growth of these cells, as in B and T cell leukaemias. Other malignancies are associated with the expression of certain proteins.
Interleukin-2 (IL-2), for example, is a cytokine of central importance to the immune system, stimulating growth and effector functions in a variety of cell types including T lymphocytes, B lymphocytes, monocytes, LAK and natural killer cells [Smith, K. A. Science 240:1169-1176 (1988); Waldmann, T. A., et al. J. Exp. Med. 160:1450-1456 (1984); Seigel, J. P., et al., Science 238:75-78 (1987); Minami, Y., et al., Ann. Rev. Immunol. 11:245-267 (1993)]. The response of peripheral blood T cells to (physiological concentrations of) IL-2 is a two stage, antigen-driven process. Antigenic stimulation of resting T cells first induces the appearance of high affinity IL-2 receptors and the production of IL-2 [Meuer, S. C., et al. Proc. Natl. Acad. Sci. USA 81:1509-1513 (1984); Cantrell, D. A. & Smith, K. A. j. Exp. Med. 158:1895-1911 (1983)]. Interaction of IL-2 with the high affinity receptor then initiates a cascade of signalling events [Minami, Y., Ann. Rev. Immunol. 11, supra; Johnston, J. A., et al., Nature 370:151-153 (1994)] which culminates in cell proliferation [Meuer, S. C., et al. Proc. Natl. Acad. Sci. USA 81:1509-1513 (1984); Smith, K. A. & Cantrell, D. A. Proc. Natl. Acad. Sci. USA 82:864-868 (1985)].
The high affinity IL-2 receptor is a heterotrimer composed of .alpha., .beta. and .gamma. chains. [Minami, Y., Ann. Rev. Immunol. 11, supra; Takeshita, I., et al. Science 257:379-382 (1992)]. The .beta. and .gamma. chains (IL-2R.beta. and IL-2R.gamma.) are constitutively expressed on resting T cells and together form a receptor of intermediate affinity [Takeshita, I., et al. Science 257:379-382 (1992); Le thi Bich-Thuy, Dukovitch, M., et al. J. Immunol. 139:1550-1556 (1987)]. Expression of the 55 kD .alpha. chain (hereinafter "IL-2R.alpha." or "Tac") and hence the high affinity receptor, occurs only transiently following engagement of the T cell receptor and is a key point of regulation in the T cell response to antigen [Smith, K. A., et al., Proc. Natl. Acad. Sci. USA 82, supra].
The overexpression of IL-2R.alpha. results in a variety of T and B cell leukaemias, most notably in HTVL-1 associated adult T cell leukaemia [Waldman, T. A., et al., Ann. Intern. Med. 116:148-160 (1992)], where it is strongly implicated in the deregulated growth of these cells. The constitutive upregulation of IL-2R.alpha. in certain T and B cell leukaemias and its association with T cell activation makes this receptor chain a natural target for suppressive immunotherapy. Therefore, the availability of an effective tool for inhibiting the expression of IL-2R.alpha. would provide a powerful reagent for gene therapy.
Therapeutic strategies to combat conditions caused by undesired expression of a protein have included the development of drugs to target the undesired proteins, means of intercellular blocking of such proteins, for example, the use of an antibody to the protein on the cell surface, and the use of drugs which will selectively kill cells expressing the undesired proteins.
For example, therapeutic strategies based on the blockade of the high affinity receptor by monoclonal antibodies (mAbs) directed against IL-2R.alpha. have been used to try to dampen a variety of other undesirable T cell-mediated reactions including allograft rejection, graft versus host disease and some forms of autoimmunity [Strom, T. B., et al., Immunological Reviews 129:131-163 (1992)]. Humanized mAbs against IL-2R.alpha. and immunotoxins such as IL-2 fused to the catalytic domain of pseudomonas exotoxin (IL-2 PE40) have also been used to selectively eradicate leukaemic or otherwise harmful T cells bearing the high affinity IL-2 receptor [Waldman, T. A., et al., Ann. Intern. Med. 116:148-160 (1992)]. However, the use of such immunotoxins and antibodies, particularly the repeated use of such antigenic molecules can elicit an immune reaction rendering their repeated use problematic.
Modem molecular techniques which inhibit the expression of specific genes provide methods for the manipulation of individual genes and cellular pathways. For example, techniques using antisense RNA, ribozymes, dominant negative mutants and targeted gene disruption have all been successfully used to inhibit the expression or function of specific genes. [Neckers, L. & Whitesell, L., Am. J. Physiol. 245:11-12 (1993); Erickson, R. P., Dev. Gene. 14:251-257 (1993); Bratty, J., et al., Biochimica et Biophysica Acta. 1261:345-359 (1993); Schaap, D., et al., J. Biol. Chem. 268:20232-20236 (1993); Capecchi, M. R., Sci. Am. 270-52-58 (1994)]. However, these methods have certain limitations. The dominant negative mutant approach, for example, requires the availability of a functionally inactive mutant which acts in a dominant manner to suppress the activity of the wild type protein. Procedures using antisense RNA produce inhibitory effects that are frequently incomplete or short lived [Neckers, L. & Whitesell, L., Am. J. Physiol. 245:11-12 (1993); Erickson, R. P., Dev. Gene. 14:251-257 (1993); Nellen, W., et al., TIBS 18:419-423 (1993)]. A major impediment to the development of effective gene inhibition protocols using antisense RNA or ribozymes is the inability to achieve a high level of expression of the inhibitor encoding DNA template in the transformed cells. This may also be a potential problem with using dominant negative mutants because of the competitive nature of the inhibition. Additionally, the targeted gene disruption technique is limited because this method cannot readily be applied to cells en masse.
It would, therefore, be desirable to have a method which can be used to achieve a high level of expression of an inhibitor to the desired molecule, specifically to IL-2 receptor proteins.
It would be desirable to have a method which can specifically target these proteins and which has wide applicability.
In U.S. patent applications Ser. Nos. 07/916,939 and 08/045,274 and PCT/US93/06735 (which is incorporated herein by reference) we discussed a method by which one can target a target molecule by the intracellular expression of antibodies ("intrabodies") for example, by targeting to specific proteins such as viral proteins, preventing their transport to the cell surface [Marasco, W. A., et al., Proc. Natl. Acad. Sci. USA 90:7889-7893 (1993)].
It would be desirable to have a method which does not introduce cytotoxic chemicals into a cell.
It would be desirable to have a method which provides a ready means of targeting undesired proteins.